The objectives of this project are to use molecular and classical approaches to investigate pathogen-arthropod interactions of vector-borne agents causing diseases of human importance in the United States. Most of our effort has concentrated on Yersinia pestis, the causative agent of bubonic plague, and spotted fever group rickettsiae. The genetic basis for Yersinia pestis blocking Oriental rat fleas was examined further. Previously we demonstrated that the hms locus, responsible for hemin storage in the outer membrane of Y. Pestis, was essential for blockage, leading to efficient transmission of the bacilli. This year the role of the 9.5 kb pesticin plasmid of Y. Pestis was also examined. An isogenic mutant of the wildtype, virulent strain 195/P was produced that lacked the 9.5 kb plasmid. This plasmid is unique to Y. Pestis, and work in other laboratories has suggested that the coagulase and fibrinolysin gene (pla) located on this plasmid may play a role in flea blockage. However, when mutant and wildtype cells were introduced into rat fleas by artificially feeding, both types of bacteria successfully blocked fleas. These data demonstrated that neither of these two phenotypes, or others encoded by this plasmid, is required for Y. pestis to block fleas. Additional work on the nonvirulent spotted fever group rickettsia infecting wood ticks. Dermacentor andersoni, on the east side of the Bitterroot Valley in Montana has shown this bacterium to be a previously undescribed species. DNA from the East Side Agent (ESA) in naturally infected tick ovarial tissue was amplified by PCR using primers based on eubacterial 16S ribosomal DNA sequences and additional sets of primers based on sequences of the citrate synthase and 190 kDa surface protein genes. Partial 16S rDNA and 190 kDa antigen gene sequences exhibited a similarity of combination of Gimenez staining, fluorescent antibody tests, PCR, and restriction fragment length polymorphism analysis, 76 of 115 female ticks collected from the east side of the Bitterroot Valley were infected. The bacterium is passed transovarially and transstadially in the ticks, and to date no vertebrate hosts have been identified. The high prevalence and stable maintenance of the ESA in wood ticks may play a role in inhibiting these ticks from also transmitting virulent spotted fever group rickettsiae.